In a number of fields, particularly the medical field, there is an emerging need to sample and manipulate extremely small volumes of material. These volumes may be in the order of one attoliter, where 1 attoliter=10−18 liter to a number of zeptoliters (1 zeptoliter=10−21 liters). For example, one may need to remove an ultrasmall volume of material from a specific location inside a biological sample such as a cell in order to perform an analysis of the constituents, for example, using a mass spectrometer. There is also need to perform an optical analysis (e.g. fluorescence) on the collected volume of material. In some cases the sampled volume can be so small (10-100 zeptoliters) that it contains on average a single analyte molecule at any given time even for high solution molar concentrations (200 μM). There may also be a need to inject small quantities of trapped material into a cell. Finally there is a need to optically trap particles such as a microscopic bead containing a drug or sensor and move it to a desired location.
Currently, small amounts of material can be picked up on the surface of sharp tipped probes. Such probes can include atomic force microscopy (AFM) probes and Near-Field Scanning Optical Microscopy (NSOM) probes. The most effective probes are those which use special coatings to enhance adherence and selectivity. Selectivity is critical to ensure that only targeted material is picked up. However these coatings add a good deal to the complexity of the procedure and also have restricted application. Tapered micropipettes can be used to extract small volumes (picoliter to femtoliter i.e., 10−12 to 10−15 liters, respectively) of material. However, it is believed that there is currently no method of extracting attoliter or smaller volumes of targeted biological material into a precision reservoir without using special coatings to enhance adherence and selectivity.
It is also difficult for micropipette probes to control the volume of extracted material. Tapered micropipette probes or pulled capillary tubing probes can be constructed to have small apertures (100 nm) and are hollow inside to accept the uptake of material. Due to the hollow nature of these probes, i.e., unrestricted and unconfined in one direction, it is difficult to extract a predetermined volume from a sample and to keep that volume intact at the end of the probe tip. These types of probes have not been successfully used to extract attoliters of material from within a biological sample. Suction techniques designed to extract material become very difficult to operate for sub-micron sized channels.
Light can be delivered down a micropipette to perform an optical analysis of biological material trapped inside the probe tip. The extracted material can also be optically probed externally using confocal or standard fluorescence microscopy techniques with lateral resolutions of ≦λ/2. However, tapered micropipettes are very inefficient at delivering light to the tip region due to their poor light guiding properties. Any fluorescence signal which returns back through such a probe would be extremely weak. It is possible to collect the fluorescence signal from the trapped material externally using confocal and other microscopies. However they require a separate and often quite sophisticated optical set-up. In addition the optical measurement can only be performed with the probe tip at or near the focus of a microscope objective and are limited to spectroscopic sampling volumes of femtoliters or larger due to the diffraction limit of light.
In another application, very small but known volumes (e.g., attoliters) of trapped material contained in a probe e.g., DNA, or a microsphere containing a drug or a sensor must be transferred to a desired location inside another material, such as the nucleus of a cell without damaging the cell. A similar problem is how to inject a small controlled volume of extracted material into a mass spectrometer for analysis. Tapered micropipettes are currently used to deliver small volumes of biological material into a cell. Tapered capillary tubes and tapered micropipettes together with electrospray techniques, for example, can be used to eject material into a mass spectrometer. Laser radiation sent down a tapered micropipette can be used to ablate material collected at the end of the tip to inject it onto a surface. However, the volume of material ejected from micropipette probes is typically in the picoliter to femtoliter range. Tapered micropipettes or capillary tubes have not been used to inject attoliter or smaller volumes of material into a cell or into a mass spectrometer.
Another problem is the trapping and manipulation, on a nanoscale of tiny particles, droplets of liquid or biological material such as cells and their transfer to a desired location. Focused light beams have been used to form optical tweezers to trap, move and release small particles as well as biological material. Optical tweezers based upon focused beams of light require precision alignment and are restricted to certain fields of use which permit access of the highly focused light beams to the particles e.g. non-turbid media, thickness of media must be less than the working distance of the lens, need for spherical aberration corrected lenses etc.